Stat6 has been implicated in interleukin (IL)-4-mediated signal transduction. To determine whether activation of Stat6 and IRS substrates occurs via related or distinct pathways, we compared the ability of IL-4 to activate Stat6 in 32D cells which do not endogenously express IRS-1 proteins with 32D cells transfected with IRS-1 or IRS-2 and found that IRS expression is not required for Stat6 activation. However, IRS expression is required for IL-4-induced c-myc expression. Structure-function analysis of the human IL-4 receptor revealed that activation of IRS and Stat6 requires a common receptor region. Although IRS and Stat6 pathways appear to be segregated, these results suggest that their activation involves common upstream factors. IL-2, IL-7, and IL-15, as well as IL-4, were demonstrated to rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human peripheral blood T cells, NK cells, and in lymphoid cell lines. In addition, JAK1 and JAK3 associated with IRS-1 and IRS-2 in T cells. Coexpression studies in COS cells demonstrate that these kinases can tyrosine phosphorylate IRS-2. These data indicate that IRS-1 and IRS-2 may have important roles in T lymphocyte activation. We studied the molecular mechanisms of the IL-4-induced prevention of apoptosis and found that IL-4 was able to protect 32D cells from apoptosis in the presence or absence of IRS-1. However, the protection from apoptosis of cells cultured in IL-4 was greater in the presence of IRS-1. The IRS-1-dependent prevention of apoptosis appears to be linked to the activation of phosphoinositol-3 kinase (PI-3K) since wortmannin and LY294002, two inhibitors of PI-3K, partially inhibited the prevention of apoptosis mediated by IL-4 in 32D-IRS-1 cells but not in 32D cells lacking IRS-1. We also found that the IRS/PI-3K pathway is, in part, responsible for the prevention of apoptosis by IL-4 in normal splenic B cell. These results demonstrate that IL-4 protects cells from apoptosis by two different pathways, one of which involves IRS-1.